Proliferation of smooth muscle cells in blood vessel walls occurs in response to vascular injury, and in association with certain disease states (Austin, G. E., et al., J Am Coll Cardiol (1985) 6:369-375). The proliferation of these cells can have negative effects due to the production of excess proteins or other matrix molecules, which, along with the cells themselves, form pathologic lesions of, for example, atherosclerosis, renal hypertension, pulmonary hypertension, vasculitis, and post-surgical vascular retinosis. These results are distinguished from the acute response to trauma characterized by blood clotting.
Glycosaminoglycans (GAG) are copolymers of alternating hexosamine and aldouronic acid residues which are found in sulfated forms and are synthesized as proteoglycans. They have collectively been called mucopolysaccharides, and those in heparin are more precisely called glycosaminoglycuronans.
To place the compositions discussed below in context, it may be noted that heparin and heparan sulfate are members of the GAG family which are classified by the nature of the hexosamine/aldouronic acid repeating units. For example, in chondroitin sulfates, the aldouronic acid is primarily D-glucuronic acid, and the hexosamine is N-acetylated 2-amino-2-deoxy-D-galactose, more commonly known as N-acetyl galactosamine and abbreviated as GalNAc.
In dermatan sulfate (chondroitin sulfate B) the aldouronic acid is mostly L-iduronic acid and the hexosamine is GalNAc. In keratan sulfate, the aldouronic acid is replaced by D-galactose, and the hexosamine is mostly N-acetylated 2-amino-2-deoxy-D-glucose, more commonly known as N-acetyl glucosamine and abbreviated as GlcNAc.
In the compositions of interest herein, heparan sulfate and heparin, the hexosamine is mostly N-acetylated or N-sulfated glucosamine (GlcN), and the aldouronic acid is mostly L-iduronic in heparin and mostly D-glucuronic acid in heparan sulfate. Heparan sulfate is commonly considered to have a higher proportion of glucuronic acid than heparin.
Problems of heterogeneity in preparations of heparan sulfate or heparin isolated from tissues make sharp distinctions difficult, since these oligosaccharides are related by the biosynthesis pathway, as explained below. Conventional heparin (used as an anticoagulant) has a molecular weight of 5-25 kDa and is extracted as a mixture of various chain lengths by conventional procedures. These procedures involve autolysis and extraction of suitable tissues, such as beef or porcine lung, intestine, or liver, and removal of other GAGs as well as nonpolysaccharide components.
The molecular weight of the chains in the extract is significantly lower than the 60-100 kd known to exist in the polysaccharide chains of the heparin proteoglycan synthesized in the tissue. The GAG moiety is synthesized bound to a peptide matrix at a serine residue through a tetrasaccharide linkage region of the sequence D-GlcA-D-Gal-D-Gal-D-Xyl.fwdarw.protein, which is then elongated at the D-GlcA residue with alternate additions of GlcNAc and GlcA.
The polysaccharide sidechains are modified by a series of enzymes which sequentially deacetylate the N-acetyl glucosamine and replace the acetyl group with sulfate, epimerize the hydroxyl at C5 of the D-glucuronic acid residue (to convert it to L-iduronic acid), sulfate the 0-2 of the resulting L-iduronic acid and the 0-6 of the glucosamine residue. Some of the chains are further sulfated at the 0-3 of the glucosamine residue, either at the heparan or heparin stage. This latter sulfation generates the active sequence required for antithrombin III binding and thus anticoagulation activity. Other chemically possible sulfation sites are on the 0-2 of D-glucuronic acid.
Due to their obvious chemical similarity, isolated "heparin" may contain considerable amounts of what might otherwise be classified as heparan sulfate.
There is an extensive body of art concerning depolymerization of heparin/heparan sulfate chains and separation of products by size. Particularly relevant is the report of Guo, Y. et al., Anal Biochem (1988) 168:54-62 which discloses the results of structure determination after the 2,5-anhydromannose at the reducing terminus is reduced to the corresponding 2,5-anhydromannitol.
The involvement of heparin or heparan sulfate or degradation products thereof in smooth muscle proliferation has been recognized for some time. Heparin and heparan sulfate can slow or arrest the vascular smooth muscle cell proliferation associated with injury described hereinabove (Clowes, A. W., et al., Nature (1977) 265:625-626). The effect of heparan sulfate and heparin on smooth muscle cell proliferation is also described by Marcum, J. A., et al. in Biology of Proteoglycan, Academic Press (1987) pp. 301-343. The inhibition of vascular smooth muscle cell growth by heparin was further described by Casteliot, J. J., Jr., et al., J Biol Chem (1982) 257:11256-11260 and the effect of heparin on vascular smooth muscle cell growth in fetal tissue was described by Benitz, W. E., et al., J Cell Physiol (1986) 127:1-7. The effect of heparin as an inhibitor of both pericyte and smooth muscle cell proliferation was shown by Orlidge, A., et al., Microvascular Research (1986) 31:41-53, and these authors further showed that chondroitin sulfate and dermatan sulfate do not have this effect. A review of the effects of heparin and heparan sulfate on the proliferation of smooth muscle cells is by Benitz, W. E. in "The Pulmonary Circulation: Normal and Abnormal", Fishman, A. P., ed., University of Pennsylvania Press (1988).
It is not clear by what mechanism these glycosaminoglycans operate, or to what extent they interact with other growth factors such as epithelial and fibroblast growth factors. It has been proposed that a 3-0 sulfate on glucosamine in an oligosaccharide of at least 5 sugars is important in this process and that both O-and N-sulfation is important (Casteliot, J. J., et al., J Cell Physiol (1984) 120:315-320; Casteliot, J. J., et al., J Cell Biol (1986) 102:1979-1984). Hexasaccharides-decasaccharides obtained from partial nitrous acid digestion of heparin bind to acidic fibroblast growth factor and aid its mitogenic activity in fibroblasts, but inhibit the proliferation of endothelial cells under some conditions (Barzu, T., et al., J Cell Physiol (1989) 140:538-548). The effective hexasaccharide was stated to have the structure: ##STR2##
Others have indicated that the presence of 2-0-sulfate glucuronic acid is not necessary for antiproliferative activity (Wright, Jr., T. C., et al., J Biol Chem (1989) 264:1534-1542). In this article, size separated fragments of defined length prepared by nitrous acid cleavage and gel filtration were further separated according to charge for some assays. Partially digested heparin separated only according to size was tested with respect to stimulation of the growth of smooth muscle cells and epithelial cells. Similar results were found in both cases, although the results were not identical. Tetrasaccharides of the type tested were shown to have very low antiproliferative activity; hexasaccharides, octasaccharides, and decasaccharides were shown to be active to approximately the same level on a weight/volume concentration basis. Also tested was a synthetic pentapeptide which represents a unique sequence of the heparin required for the binding of heparin to antithrombin III; this pentapeptide was active in inhibiting proliferation for smooth muscle but not epithelial cells. The size-separated fractions were then treated chemically to produce "O-oversulfation" and this treatment enhanced the inhibitory activity; indeed, oversulfation of the tetrasaccharide fragment preparation yielded a tetrasaccharide fraction which was active in inhibiting proliferation. The converse process, comprising desulfation and reacetylation of the amino groups of glucosamine results in a reduction in antiproliferative activity. These fragments could, however, be made more active by subsequent oversulfation.
Also capable of reducing the activity of the heparin fragments was reduction of the carboxyl groups so as to reduce the total negative charge. O-oversulfation partially restores this activity. These results with N-desulfated, N-acetylated fragments which are lacking in antiproliferative activity is distinguishable from previous results wherein similarly treated heparin retains the capacity to prevent cell division because of the size dependency of the antiproliferative activity-larger fragments being more powerful in general than smaller ones.
When the size separated fraction was further fractionated according to charge, it was found that the most highly charged fractions showed the greatest activity. Furthermore, it was shown that although the synthetic pentasaccharide identified as the antithrombin III binding site is capable of inhibiting proliferation in smooth muscle cells, any treatment of heparin which would destroy the sequence corresponding to this pentapeptide (i.e., periodate treatment) does not destroy antiproliferative activity.
Methods of synthesizing oligosaccharides are disclosed in U.S. Pat. No. 4,943,630 issued Jul. 14, 1990 which is incorporated herein by reference to disclose such methods.
The present inventors have now found that an enhanced antiproliferative activity with respect to smooth muscle cells is associated with an oligosaccharide portion of the heparin or heparan sulfate GAGs which is highly sulfated and contains 6 or 8 saccharide units and have provided synthesis mechanisms for making polysaccharides containing 6 or more sugar residues, which oligosaccharides have enhanced antiproliferative activity with respect to smooth muscle cells.